Bottom Line:
We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes.Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system.In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

Affiliation: Department of Cell and Developmental Biology, Biocenter of the University of Würzburg, D-97074 Würzburg, Germany.

ABSTRACTNuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

Figure 1: eIF-5A is required for nuclear export of GST–Rev–NES and Rev-mediated RNA export, but not for CTE RNA and GST–PKI–NES export in Xenopus oocytes. (A) To analyze interaction of the Rev–NES with eIF-5A in solution, glutathione–Sepharose beads coupled to GST–Rev (lanes 1 and 1′) or, as a control, GST (lanes 2 and 2′) were incubated with the extract of 150 Xenopus oocytes, pelleted, and then processed for immunoblotting with antibodies to eIF-5A. Bound proteins are shown in lanes 1 and 2, and the unbound material are shown in lanes 1′ and 2′. eIF-5A binds specifically to GST–Rev–NES (compare lanes 1 and 1′). Molecular mass standards are indicated in kD. (B) Oocyte injection protocol. After injection and incubation, oocytes were manually dissected in nuclear (N) and cytoplasmic (C) fractions. (C) Proteins were separated by 18% SDS-PAGE, and blots were probed with antibodies against GST and BSA. In the presence of control IgG, within 2 h, most GST–Rev–NES migrates from the nucleus to the cytoplasm (lanes 1 and 2). In contrast, in the presence of antibodies to eIF-5A, the nuclear export of GST–Rev–NES is largely inhibited (lanes 3 and 4). Coinjection of wt eIF-5A protein overcomes the blocking effect of anti–eIF-5A antibodies on GST–Rev–NES export (lanes 5 and 6). To monitor the site of injection, the export substrate was injected along with BSA, which is not exported. The exclusive presence of BSA in the nuclei confirms the specificity of the nuclear injections (lanes 1, 3, and 5). Molecular mass standards are indicated in kD. (D and E) RNA was analyzed by 8 M 6% acrylamide urea gel electrophoresis followed by autoradiography. (D) In the presence of Rev, the RRE RNA is exported (lane 2). In contrast, antibodies against eIF-5A inhibit this export (lane 4). (E) Antibodies against eIF-5A do not prevent the nuclear export of CTE RNA (lanes 2 and 4), indicating that eIF-5A is not involved in the CTE RNA export pathway. Nuclear injected CTE M2/M11 RNA mutant remains in the nuclei (lanes 1 and 3). The nuclear localization of U6-RNA confirms the accuracy of nuclear injections. (F) Nuclear export of the injected GST–PKI–NES fusion protein was analyzed as shown in B. The export of GST–PKI–NES was unaffected by the presence of anti–eIF-5A antibodies (lanes 2 and 4), confirming that eIF-5A is not involved in PKI-NES–mediated nuclear export.

Mentions:
The Rev–NES has been shown previously to interact with HeLa cell–derived and recombinant eIF-5A (Ruhl et al. 1993; Bevec et al. 1996). To verify the interaction of the Rev–NES with oocyte-derived eIF-5A, we first performed binding studies in solution. For this, purified GST–Rev–NES fusion protein was immobilized on glutathione–Sepharose beads and incubated with total protein extracts from Xenopus oocytes. The beads were then pelleted by centrifugation, and the bound and unbound material was analyzed by Western analysis using antibodies directed against eIF-5A (Elfgang et al. 1999). As shown, eIF-5A bound the GST–Rev–NES fusion protein (Fig. 1 A, compare lanes 1 and 1′) but not GST alone (Fig. 1 A, compare lanes 2 and 2′).

Figure 1: eIF-5A is required for nuclear export of GST–Rev–NES and Rev-mediated RNA export, but not for CTE RNA and GST–PKI–NES export in Xenopus oocytes. (A) To analyze interaction of the Rev–NES with eIF-5A in solution, glutathione–Sepharose beads coupled to GST–Rev (lanes 1 and 1′) or, as a control, GST (lanes 2 and 2′) were incubated with the extract of 150 Xenopus oocytes, pelleted, and then processed for immunoblotting with antibodies to eIF-5A. Bound proteins are shown in lanes 1 and 2, and the unbound material are shown in lanes 1′ and 2′. eIF-5A binds specifically to GST–Rev–NES (compare lanes 1 and 1′). Molecular mass standards are indicated in kD. (B) Oocyte injection protocol. After injection and incubation, oocytes were manually dissected in nuclear (N) and cytoplasmic (C) fractions. (C) Proteins were separated by 18% SDS-PAGE, and blots were probed with antibodies against GST and BSA. In the presence of control IgG, within 2 h, most GST–Rev–NES migrates from the nucleus to the cytoplasm (lanes 1 and 2). In contrast, in the presence of antibodies to eIF-5A, the nuclear export of GST–Rev–NES is largely inhibited (lanes 3 and 4). Coinjection of wt eIF-5A protein overcomes the blocking effect of anti–eIF-5A antibodies on GST–Rev–NES export (lanes 5 and 6). To monitor the site of injection, the export substrate was injected along with BSA, which is not exported. The exclusive presence of BSA in the nuclei confirms the specificity of the nuclear injections (lanes 1, 3, and 5). Molecular mass standards are indicated in kD. (D and E) RNA was analyzed by 8 M 6% acrylamide urea gel electrophoresis followed by autoradiography. (D) In the presence of Rev, the RRE RNA is exported (lane 2). In contrast, antibodies against eIF-5A inhibit this export (lane 4). (E) Antibodies against eIF-5A do not prevent the nuclear export of CTE RNA (lanes 2 and 4), indicating that eIF-5A is not involved in the CTE RNA export pathway. Nuclear injected CTE M2/M11 RNA mutant remains in the nuclei (lanes 1 and 3). The nuclear localization of U6-RNA confirms the accuracy of nuclear injections. (F) Nuclear export of the injected GST–PKI–NES fusion protein was analyzed as shown in B. The export of GST–PKI–NES was unaffected by the presence of anti–eIF-5A antibodies (lanes 2 and 4), confirming that eIF-5A is not involved in PKI-NES–mediated nuclear export.

Mentions:
The Rev–NES has been shown previously to interact with HeLa cell–derived and recombinant eIF-5A (Ruhl et al. 1993; Bevec et al. 1996). To verify the interaction of the Rev–NES with oocyte-derived eIF-5A, we first performed binding studies in solution. For this, purified GST–Rev–NES fusion protein was immobilized on glutathione–Sepharose beads and incubated with total protein extracts from Xenopus oocytes. The beads were then pelleted by centrifugation, and the bound and unbound material was analyzed by Western analysis using antibodies directed against eIF-5A (Elfgang et al. 1999). As shown, eIF-5A bound the GST–Rev–NES fusion protein (Fig. 1 A, compare lanes 1 and 1′) but not GST alone (Fig. 1 A, compare lanes 2 and 2′).

Bottom Line:
We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes.Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system.In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

Affiliation:
Department of Cell and Developmental Biology, Biocenter of the University of Würzburg, D-97074 Würzburg, Germany.

ABSTRACTNuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).